Nucleic acid hybridization assays are commonly used in genetic research, biomedical research and clinical diagnostics. In a basic nucleic acid hybridization assay, the nucleic acid of interest is hybridized, in single-stranded form, to a labeled single-stranded nucleic acid probe and resulting labeled duplexes are detected. Variations of this basic scheme have been developed to enhance accuracy, facilitate the separation of the duplexes to be detected from extraneous materials, and/or amplify the signal that is detected.
Commonly assigned U.S. patent application Ser. No. 07/559,961, filed Jul. 27, 1990, now U.S. Pat. No. 5,430,136 and incorporated by reference herein, describes a technique whereby selectably cleavable sites are introduced into oligonucleotide chains, enabling release of a detectable label after hybridization is complete. As explained in that application, selectably cleavable sites are useful in a number of different types of hybridization assay formats. For example, in one type of assay in which hybridization gives rise to a solid-supported duplex of a labeled probe and sample DNA, a selectably cleavable site contained within the hybrid structure will enable ready separation of the label from the solid support. Commonly assigned U.S. Pat. Nos. 4,775,619 and 5,118,605 are respectively directed to the use of restriction endonuclease cleavable sites in such assays and the use of chemically cleavable sites (e.g., disulfide linkages, 1,2-diols, and the like). These cleavable sites can be introduced during oligonucleotide synthesis, and are cleavable with restriction endonucleases in the case of restriction sites and with particular chemical reagents, e.g., with thiols, periodate, or the like, in the case of chemically cleavable sites.
The present invention is also directed in part to the incorporation of selectably cleavable sites into polynucleotides. The cleavable sites herein are contained within a linker arm present at the 1 position of a deoxyribose molecule. In addition to providing such cleavable sites, the invention also relates to the creation of "abasic sites" within polynucleotides, i.e., monomeric units which contain the deoxyribose ring but do not have a purine or pyrimidine base present at the 1 position. Such abasic sites are useful in a wide variety of contexts, as will be explained in detail hereinbelow. For example, an abasic site may be used to create branched DNA, i.e., a multimeric polynucleotide structure in which three polynucleotide chains emanate from a single deoxyribose unit. These branch points are extremely useful in providing large, "multimeric" DNA structures which can then be used in amplification assays. Abasic sites may also be used in other ways, e.g., in the synthesis of DNA bound to a solid support (typically although not necessarily at the 1 position), to reverse the direction of chemical DNA synthesis, i.e., 3'.fwdarw.5' to 5'.fwdarw.3' or vice versa, and in triple helix formation.
Thus, in addition to utility in providing cleavable sites within oligonucleotide or polynucleotide chains, the invention enables a number of procedures deriving from the presence of linker arms at the 1 position of a monomeric deoxyribose unit rather than purine or pyrimidine bases as present in conventional nucleotide structures.